Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.

Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.     

Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with heme

Kinetics of the reconstitution of hemoglobin from semihemoglobins and with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins and are occupied by heme in the and chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin , the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin . Correspondence to: Y. Kawamura-Konishi     

Double-Blind Study of the Chronopharmacotherapy of Depression

To investigate the possibility of chronotherapy with antidepressants for patients with depression, we gave single daily doses of clomipramine 150 mg/day to 30 patients with depression at three different times of day, i.e., morning, noon, or before bedtime, using a double-blind method over a 4-week period. Beneficial effects differed according to the administration time of day, with the most effective result being found for the administration at noon. Time-dependent differences in side effects were observed in tremors and dryness of mouth. An additional 10 patients were administered their medications three times a day by traditional, equally divided doses, and the efficacy was inferior than daily single doses at noon. The study results showed the significance of the administration time of day for the benefits and side effects of antidepressant therapy for depression.     

Scyliorhinus tokubee sp. nov. from Izu Peninsula,southern Japan (Scyliorhinidae,Elasmobranchii)

A new catshark,Scyliorhinus tokubee sp. nov., is described based on specimens from the coast of Shirahama, eastern Izu Peninsula, southern Japan. The present species is distinguished from other congeners in having a particular coloration with dark saddles, blotches and numerous small light spots, a wide oral cleft, the anterior nasal flap not reaching the oral cleft, a short interspace between the dorsal fins, developed clasper hooks, and some meristic characters (number of vertebrae, jaw teeth, and spiral valve turns). This species has been bred under captivity for several years in Shimoda Floating Aquarium.     

Immunoaffinity purification of type I protein kinase C

We designed a simple procedure for the purification of type I protein kinase C, using immunoaffinity chromatography with a monoclonal antibody, MC-1b, obtained by rescreening hybridoma cells available for an affinity ligand. Western blotting demonstrated that MC-1b specifically reacted with type I protein kinase C, and the enzyme molecule dissociated from MC-1b-coupled Sepharose 4B with mild eluants such as thiocyanate retained the kinase activity. A 1148-fold purification was achieved and 210 micrograms of type I protein kinase C was obtained from three rabbit brains, by means of a two-step procedure, using DEAE-cellulose and immunoaffinity chromatography. The resultant preparation was homogeneous, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis hydroxylapatite chromatography, and immunological analysis using MC-1a, MC-2a, and MC-3a.     

Generality of the action of various maturation-promoting factors

It has been known in amphibians and starfishes that a cytoplasmic factor called maturation-promoting factor (MPF), produced in maturing oocytes under the influence of the maturation-inducing hormones, can induce germinal vesicle breakdown (GVBD) and the subsequent process of meiotic maturation. The present study revealed that injection of cytoplasm of maturing starfish oocytes (starfish MPF) into immature sea cucumber oocytes brought about maturation of the recipients. Amphibian MPF obtained from mature oocytes of Xenopus laevis or Bufo bufo was found to induce maturation of starfish oocytes following injection. Cytoplasm taken from cleaving starfish blastomeres induced maturation when injected into immature starfish oocytes. The maturation-inducing activity of cytoplasm of starfish blastomeres changed along with the mitotic cell cycle during 1- to 4-cell stages so far tested and reached a peak just before cleaving. Furthermore, an extract of mammalian cultured cells, CHO or V-79, synchronized in M phase, induced GVBD in starfish oocytes following injection, whereas S phase extract had little activity. These facts suggest that MPF generally brings about nuclear membrane breakdown in both meiosis and mitosis, and that the nature of MPF is very similar among vertebrates and invertebrates.     

A granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by carrageenin-induced inflammatory cells of mice

Seven days after subcutaneous injection of 2% carrageenin solution in mice, the induced inflammatory tissue was isolated and treated with 0.1% trypsin. When the dispersed cells were incubated in a nutrient medium containing 5--20% calf serum, the cells grew adhering to the culture-dish surface and colony-stimulating factor (CSF) was accumulated in the medium gradually. Addition of lipopolysaccharide (Escherichia coli endotoxin) in the cell culture did not enhance the CSF production. It was shown by isoelectrofocusing that the majority of the produced CSF was an acidic molecule (pI = 3.8), while the treatment of this CSF with neuraminidase yielded a less acidic CSF species (pI = 5.1). Upon gel-filtration chromatography in the presence of 6 M guanidine HCl, the CSF exhibited an apparent molecular weight of 42,000. On the other hand, polyacrylamide gel-electrophoresis in the presence of 0.1% sodium dodecyl sulfate gave a molecular weight estimate of 65,000. Microscopic examination of the bone marrow cell culture showed that about one-third of the colonies were granulocytic. Addition of prostaglandin E₁(PGE₁) in the bone marrow cell culture significantly inhibited the action of the CSF, while prostaglandin D₂ was less inhibitory than PGE₁. The result suggests that the cells isolated from the inflammatory tissue are capable of producing CSF, which may have a role for proliferation and maturation of the mononuclear phagocytes at the site of inflammation.     

Plasma levels of 16 alpha-hydroxypregnenolone, 16 alpha-hydroxyprogesterone and 16 alpha-hydroxydehydroepiandrosterone in the fetus and neonates.

Simultaneous determination of unconjugated 16 alpha-hydroxypregnenolone (16 alphaOH-Preg), 16 alpha-hydroxyprogesterone (16 alphaOH-Prog) and 16 alpha-hydroxydehydroepiandrosterone (16 alphaOH-DHEA) in fetal and neonatal plasma was performed utilizing a newly developed radioimmunoassay. In all neonates, the three 16 alpha-hydroxysteroid levels were consistently higher in umbilical cord plasma than in the maternal peripheral circulation. 16 alpha-OH-Preg in the umbilical arterial plasma increased from 11.2 +/- 3.1 at 24 weeks to 29.7 +/- 12.0 ng/ml at term, 16 alphaOH-Prog from 15.5 +/- 3.2 to 34.3 +/- 11.0 ng/ml and 16 alphaOH-DHEA from 5.1 +/- 1.2 to 5.9 +/- 1.0 ng/ml. In the anencephalic neonates, only 16 alphaOH-Preg showed an increase pattern under ACTH priming. 16 alpha-OH-Preg levels for normal full term neonates remain relatively constant at the first 24 hr and show a slight decrease at 3 days post partum. In small full term neonates, 16 alphaOH-Preg levels in umbilical arterial plasma are considerably higher than in normal neonates and remain at roughly equivalent levels for the first 5 days post partum. 16 alphaOH-Prog and 16 alphaOH-DHEA levels in umbilical arterial plasma in normal and small full term neonates are almost equal and both groups show a rapid decrease during the first 24 hr. Comparison with findings of the three 16 alpha-hydroxysteroids in fetal and neonatal plasma is discussed.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.